Immunologic characteristics of human gingival fibroblasts in response to oral bacteria.

BACKGROUND AND OBJECTIVE: There is ample evidence that gingival fibroblasts (GFs) participate in the immune response to oral bacteria and serve as immune-regulatory cells. The objective of this study was to investigate the innate immune response of GFs to oral bacteria. MATERIAL AND METHODS: Human GFs were cocultured with relatively less-pathogenic (Leptotrichia wadei, Fusobacterium nucleatum and Campylobacter gracilis) and pathogenic red-complex bacteria. The expression of mRNA for antimicrobial peptides [AMPs; namely human beta defensins (HBDs)], chemokines with antimicrobial activity [chemokine C-X-C motif (CXCL)10, CXCL11 and chemokine C-C motif ligand 20 (CCL20)] and proinflammatory mediators [interleukin (IL)6 and IL8] and the levels of CXCL11, CCL20, IL-6 and IL-8 accumulated in supernatants were analyzed using real-time PCR and ELISA, respectively. The proteolytic activities of CXCL11, CCL20, IL-6 and IL-8 produced by six species of bacteria were also determined. RESULTS: The relatively less-pathogenic bacteria strongly up-regulated the expression of antimicrobial chemokines and proinflammatory mediators, whereas the red-complex bacteria stimulated low levels, or often suppressed, expression of these factors. Regarding the regulation of AMPs, the inhibition of HBD3, HBD106 and HBD107 mRNAs by Porphyromonas gingivalis was noticeable; however, differences between the two bacterial groups were not conspicuous. Differential degradation of proteins by the six bacterial species was observed: P. gingivalis and Treponema denticola degraded proteins well, whereas the other species degraded proteins to a relatively lower degree. CONCLUSION: The invasion of red-complex bacteria into gingival connective tissue can suppress the immune response of GFs and can be a source of persistent infection in connective tissue.

Lactic acidosis not hyperlactatemia as a predictor of in hospital mortality in septic emergency patients.

OBJECTIVE: To compare the prognostic abilities of lactic acidosis and hyperlactatemia and determine the influence of metabolic acidosis when attempting to predict the outcome of septic patients using serum lactate levels. METHOD: 126 patients with severe sepsis or septic shock were prospectively included in this study at an emergency department. Haemodynamic variables, simplified acute physiology score (SAPS) II, arterial blood gas studies and serum lactate levels were obtained at the time of presentation (0 h) and 4 h after presentation. The probability of mortality was calculated using SAPS II scores and compared with actual inhospital mortality at different serum lactate levels and arterial pH. Survival curves for lactic acidosis and severe hyperlactatemia were analysed using the Kaplan-Meier method. RESULTS: The calculated probability of mortality decreased from 35.7% (95% CI 30.2 to 41.2) at presentation to 29.3% (95% CI 24.2 to 34.4) at 4 h post-presentation. 27.0% of patients (34) died in hospital. Arterial pH and SAPS II scores were independent factors for predicting mortality of septic patients, at 0 h and 4 h. Serum lactate levels were closely related to severity of illness and metabolic acidosis in septic patients. Patients with lactic acidosis had significantly higher inhospital mortality than patients with normal pH and normal lactate levels by Kaplan-Meier survival analysis as determined based on measurements made at 0 h and 4 h (p<0.001, p<0.001 by the log-rank test, respectively). No significant difference in survival was found between patients with hyperlactatemia and those with normal pH and serum lactate levels. CONCLUSION: Lactic acidosis not hyperlactatemia was found to predict inhospital mortality more exactly in severe sepsis and septic shock patients. The acid-base state should be considered when attempting to predict the outcome of septic patients using serum lactate levels.

Applications of the pulsatile flow versatile ECLS: in vivo studies.

INTRODUCTION: T-PLS (Twin-Pulse Life Support) is the first commercial pulsatile ECLS (Extra Corporeal Life Support) device (1). The dual sac structure of T-PLS can effectively reduce high membrane oxygenator inlet pressure and hemolysis. To verify both the use of T-PLS for ECLS and the advantages of T-PLS, we tested various models. METHOD AND RESULTS: In the partial CPB (cardio pulmonary bypass) model (swine), T-PLS (N = 6), and Biopump (N = 2), a single pulsatile pump (N = 2), were compared. In the case of single pulsatile flow, during pump systole, pressure increased to 700 - 800 mmHg at the inlet port of the membrane oxygenator. fHb, a hemolysis measurement value, was about 80 mg/dL at 3 hours. On the contrary, because of T-PLS's dual sac system, the pressure of T-PLS had a maximum value of about 250 mmHg and fHb was similar to that of the commercial centrifugal pumps. In the total CPB model (bovine, N = 6), the heart was stopped via cardioplegia (Kcl). T-PLS flow was maintained at 3.0-4.5 L/min. T-PLS functioned like a natural heart, having a pulse pressure of 26-43 mmHg and a pulse rate of 40-60 bpm (beats per minute). In the emergency case model (canine, N = 6), T-PLS was started 10 minutes after cardiac arrest from electronic shock. In spite of cardiac arrest for a period of 40 minutes, the heart was recovered after defibrillation. In the ARDS (Acute Respiratory Distress Syndrome) model (canine, N = 6), minimal ventilator parameters were set: tidal volume 130 ml, respiration rate = bpm, FiO2 = 10%. Three hours after starting T-PLS, PO2 of the carotid artery blood (after 2 hours: 195 +/- 89.4; after 3 hours: 258 +/- 99.3 mmHg) was above half the value of the femoral artery but was within normal range. CONCLUSION: It is suggested that a portable pulsatile ECLS like T-PLS may be used as a CPB device and as an alternative CPR (cardiopulmonary resuscitation) device in the case of cardiac arrest. Due to the pulsatile flow, oxygenated blood is delivered to the patient without overloading the ARDS patients heart.

Immunostimulatory DNA sequences inhibit respiratory syncytial viral load, airway inflammation, and mucus secretion.

BACKGROUND: Immunostimulatory DNA sequences (ISS) activate the innate immune system to generate antiviral cytokines, such as IFN-gamma. OBJECTIVE: This study investigated whether ISS could reduce viral load, mucus secretion, airway inflammation, and airway hyperreactivity to methacholine in a mouse model of respiratory syncytial virus (RSV) infection. METHODS: Mice were pretreated with ISS 6 days before RSV infection, and lung indices of RSV viral load (viral titer and PCR), bronchoalveolar lavage fluid cytokines (IFN-gamma), airway inflammation (peribronchial inflammation and periodic acid-Schiff-positive mucus cells), and airway hyperreactivity (methacholine responsiveness) were assessed 4 to 6 days after RSV infection. RESULTS: ISS induced the expression of the antiviral cytokine IFN-gamma in the lung, and this was associated with significantly reduced RSV viral titers, mucus secretion, and peribronchial inflammation. ISS reduced, but did not significantly inhibit, RSV-induced airway hyperreactivity to methacholine. CONCLUSION: Because ISS induced significant levels of lung IFN-gamma, an immunization strategy based solely on the administration of IFN-gamma may be insufficient to inhibit RSV-induced airway hyperreactivity to methacholine, an endpoint important in the subset of RSV-infected subjects with asthma.

Involvement of p38 mitogen-activated protein kinase and apoptosis signal-regulating kinase-1 in nitric oxide-induced cell death in PC12 cells.

Although nitric oxide (NO) plays key signaling roles in the nervous systems, excess NO leads to cell death. In this study, the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and apoptosis signal-regulating kinase-1 (ASK1) in NO-induced cell death was investigated in PC12 cells. NO donor transiently activated p38 MAPK in the wild type parental PC12 cells, whereas the p38 MAPK activation was abolished in NO-resistant PC12 cells (PC 12-NO-R). p38 MAPK inhibitors protected the cells against NO-induced death, whereas the inhibitors were not significantly protective against the cytotoxicity of reactive oxygen species. Stable transfection with dominant negative p38 MAPK mutant reduced NO-induced cell death. Stable transfection with dominant negative mutant of ASK1 attenuated NO-stimulated activation of p38 MAPK and decreased NO-induced cell death. These results suggest that p38 MAPK and its upstream regulator ASK1 are involved in NO-induced PC12 cell death.

Distinct characteristic of Galpha(h) (transglutaminase II) by compartment: GTPase and transglutaminase activities.

Galpha(h) (transglutaminase II) is a bifunctional enzyme possessing transglutaminase and GTPase activities. To better understand the factors affecting these two functions of Galpha(h), we have examined the characteristics of purified Galpha(h) from membrane and cytosol. GTP binding activity of mouse heart Galpha(h) was higher in membrane than that from cytosol. Furthermore, phospholipase C-delta1 (PLC-delta1) activity and coimmunoprecipitation of Galpha(h)-coupled PLC-delta1 in the alpha(1)-adrenoceptor-Galpha(h)-PLC-delta1 complex preparations were increased by phenylephrine in the presence of membranous Galpha(h). On the other hand, transglutaminase activity of cytosolic Galpha(h) was higher than that from membrane Galpha(h). These results demonstrate that bifunctions of Galpha(h) are regulated by its localization that can reflect the cellular functions of Galpha(h).

Phospholipase Cdelta1 is a guanine nucleotide exchanging factor for transglutaminase II (Galpha h) and promotes alpha 1B-adrenoreceptor-mediated GTP binding and intracellular calcium release.

Effectors involved in G protein-coupled receptor signaling modulate activity of GTPases through GTPase-activating protein or guanine nucleotide exchanging factor (GEF). Phospholipase Cdelta1 (PLCdelta1) is an effector in tissue transglutaminase (TGII)-mediated alpha1B-adrenoreceptor (alpha(1B)AR) signaling. We investigated whether PLCdelta1 modulates TGII activity. PLCdelta1 stimulated GDP release from TGII in a concentration-dependent manner, resulting in an increase in GTPgammaS binding to TGII. PLCdelta1 also inhibited GTP hydrolysis by TGII that was independent from the alpha(1B)AR. These results indicate that PLCdelta1 is GEF for TGII and stabilizes the GTP.TGII complex. When GEF function of PLCdelta1 was compared with that of the alpha(1B)AR, the alpha(1B)AR-mediated GTPgammaS binding to TGII was greater than PLCdelta1-mediated binding and was accelerated in the presence of PLCdelta1. Thus, the alpha(1B)AR is the prime GEF for TGII, and GEF activity of PLCdelta1 promotes coupling efficacy of this signaling system. Overexpression of TGII and its mutants with and without PLCdelta1 resulted in an increase in alpha(1B)AR-stimulated Ca2+ release from intracellular stores in a TGII-specific manner. We conclude that PLCdelta1 assists the alpha(1B)AR function through its GEF action and is primarily activated by the coupling of TGII to the cognate receptors.

Phospholipase C-delta1 is activated by capacitative calcium entry that follows phospholipase C-beta activation upon bradykinin stimulation.

To characterize the regulatory mechanism of phospholipase C-delta1 (PLC-delta1) in the bradykinin (BK) receptor-mediated signaling pathway, we used a clone of PC12 cells, which stably overexpress PLC-delta1 (PC12-D1). Stimulation with BK induced a significantly higher Ca(2+) elevation and inositol 1,4,5-trisphosphate (IP(3)) production with a much lower half-maximal effective concentration (EC(50)) of BK in PC12-D1 cells than in wild type (PC12-W) or vector-transfected (PC12-V) cells. However, BK-induced intracellular Ca(2+) release and IP(3) generation was similar between PC12-V and PC12-D1 cells in the absence of extracellular Ca(2+), suggesting that the availability of extracellular Ca(2+) is essential to the activation of PLC-delta1. When PC12-D1 cells were treated with agents that induce Ca(2+) influx, more IP(3) was produced, suggesting that the Ca(2+) entry induces IP(3) production in PC12-D1 cells. Furthermore, the additional IP(3) production after BK-induced capacitative calcium entry was detected in PC12-D1 cells, suggesting that PLC-delta1 is mainly activated by capacitative calcium entry. When cells were stimulated with BK in the presence of extracellular Ca(2+), [(3)H]norepinephrine secretion was much greater from PC12-D1 cells than from PC12-V cells. Our results suggest that PLC-delta1 is activated by capacitative calcium entry following the activation of PLC-beta, additively inducing IP(3) production and Ca(2+) rise in BK-stimulated PC12 cells.

Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as an effector.

Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified. Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghalpha is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem. J. 315, 739-744]. To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor-Ghalpha-PLC-delta1 complex. PLC-delta1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghalpha (GTP-bound Ghalpha). Furthermore the stimulated PLC-delta1 activity resulting from activation of Ghalpha via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP. Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-delta1 in the three-component preparation by anti-Gh7alpha antibody resulted in the PLC-delta1 being tightly coupled to activated Ghalpha on stimulation of the oxytocin receptor. These results indicate that PLC-delta1 is the effector for Ghalpha-mediated oxytocin receptor signalling.

Identification of a distinct molecular mass G alpha(h) (transglutaminase II) coupled to alpha1-adrenoceptor in mouse heart.

Our previous studies on alpha1-adrenoceptor signaling suggested that G alpha(h) family is a signal mediator in different species. To elucidate the species-specificity of G alpha(h) family in molecular mass, we used the solubilized membranes from mouse heart and the ternary complex preparations containing alpha1-agonist/receptor/G-protein. Binding of [35S]GTPgammaS and the intensity of the [alpha-32P]GTP photoaffinity labeled protein resulting from activation of the alpha1-adrenoceptor were significantly attenuated by the antagonist, phentolamine. The molecular mass of the specific GTP-binding protein was approximately 72-kDa; homologous with G alpha(h) (transglutaminase II) family. Furthermore, immunological cross-reactivity of ternary complex from mouse heart and purified G alpha(h) from rat, guinea pig, and bovine using anti-G alpha(h7) antibody showed that their molecular masses were distinctly different and approximately 72-kDa G alpha(h) from mouse heart was the lowest molecular mass. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the alpha1-adrenoceptor in the ternary complex preparation by anti-G alpha(h7) antibody, the G alpha(h) family protein tightly coupled to alpha1-adrenoceptor. These results demonstrate the species-specificity of G alpha(h) family in molecular mass, especially the lowest molecular mass in mouse.

A new member of alpha 1-adrenoceptor-coupled G alpha h (transglutaminase II) family in pig heart: purification and characterization.

We previously reported an identification of a 77-kDa GTP-binding protein that co-purified with the alpha 1-adrenoceptor following ternary complex formation. In the present paper, we report on the purification and characterization of this GTP-binding protein (termed G alpha h5) isolated from pig heart membranes. After solubilization of pig heart membranes with NaCl, G alpha h5 was purified by sequential chromatographies using DEAE-Cellulose, Q-Sepharose, and GTP-agarose columns. The protein displayed high-affinity GTP gamma S binding which is Mg(2+)-dependent and saturable. The relative order of affinity of nucleotide binding by G alpha h5 was GTP > GDP > ITP >> ATP > or = adenyl-5'-yl imidodiphosphate, which was similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, the G alpha h5 demonstrated transglutaminase (TGase) activity that was blocked either by EGTA or GTP gamma S. In support of these observations, the G alpha h5 was recognized by a specific antibody to G alpha h7 or TGase II, indicating a homology with G alpha h (TGase II) family. These results demonstrate that 77-kDa G alpha h5 from pig heart is an alpha 1-adrenoceptor-coupled G alpha h (TGase II) family which has species-specificity in molecular mass.

Modulation of large conductance Ca2+-activated K+ channel by Galphah (transglutaminase II) in the vascular smooth muscle cell.

Among G-proteins, Gh is unique in structural differences in the GTP-binding domain and possessing transglutaminase activity. We have studied the role of G protein in modulation of large conductance Ca2+-activated K+ (Maxi-K+) channel by the inside-out mode of patch clamp in smooth muscle cells from superior mesenteric artery of the rabbit. When the non-hydrolyzable GTP analogue, GTPgammaS, was applied, the channel activity was increased about 2.5-fold. Addition of GDPbetaS resulted in reversal of the GTPgammaS effect. When the Galphah7 antibody was applied, the GTPgammaS-stimulated channel activity was significantly inhibited to control level, suggesting that Galphah is involved in activation of the Maxi-K+ channel in smooth muscle cells.

Involvement of superoxide radical in the impaired endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbits.

Involvement of the superoxide radical in impaired relaxation of penile cavernous smooth muscle in hypercholesterolemia was investigated. New Zealand White rabbits (n = 40) were randomly divided into control and treatment groups. The control group (n = 20) received a regular diet while the treatment group (n = 20) was fed a diet of 2% cholesterol for 8 weeks. Blood level of cholesterol in the cholesterol-fed group was significantly higher than that of the control group. The contraction responses of cavernous tissues to norepinephrine were not significantly different in the two groups. The relaxation responses to endothelium-dependent agents (acetylcholine, bradykinin) were significantly reduced in the hypercholesterolemic group compared with the control group. However, the relaxation responses to endothelium-independent agents (papaverine, verapamil) were not significantly different in the two groups. The production of superoxide radicals was significantly higher in the hypercholesterolemic group than in the control group (P < 0.01). The activity of superoxide dismutase (total SOD, Mn-SOD, Cu,Zn-SOD) increased significantly in the hypercholesterolemic group compared with the control group (P < 0.05). The activities of catalase and glutathione peroxidase also increased in the hypercholesterolemic group, but were not significantly higher than those of the control group. Therefore, production of the superoxide radicals in rabbit cavernous tissues increases in the state of hypercholesterolemia, which may lead to functional impairment of cavernous smooth muscle relaxation in response to endothelium-mediated stimuli.

Oxytocin receptor couples to the 80 kDa Gh alpha family protein in human myometrium.

One of the primary functions of the oxytocin receptor is to modulate intracellular calcium levels in myometrium. The oxytocin receptor has been purified and cloned. Although it has been suggested that oxytocin receptor couples with a GTP-binding regulatory protein (G-protein), the identity of this G-protein remains unclear. To elucidate the mechanism of oxytocin receptor signalling, we used the oxytocin-receptor-G-protein ternary complex preparation from human myometrium, and evaluated oxytocin-mediated activation of [35S]guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) binding and [alpha-32P]GTP photoaffinity labelling to a G-protein. Binding of [35S]GTP[S] and the intensity of the [alpha-32P]GTP photoaffinity labelled protein resulting from activation of the oxytocin receptor were significantly attenuated by the selective oxytocin antagonist, desGlyNH2d(CH2)5[Tyr(Me)2,Thr4]OVT. Furthermore, the molecular mass of the specific GTP-binding protein was approximately 80 kDa; homologous with the Gh alpha family, the new class of GTP-binding proteins first identified in rat liver that couples to the alpha 1B-adrenoceptor. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the oxytocin receptor in the ternary complex preparation by anti-Gh7 alpha antibody, the Gh alpha family protein tightly coupled to the oxytocin receptor. These findings demonstrate that oxytocin receptor couples with approximately 80 kDa Gh alpha in signal mediation.

A 50 KDa protein modulates guanine nucleotide binding of transglutaminase II.

Regulation of cellular response is an important mechanism for controlling cellular functions. The transmembrane signaling of the hormone receptors is regulated by GTP-binding proteins (GTPases) and their associated proteins. Our previous studies demonstrated that the bifunctional GTP-binding protein, G alpha h (transglutaminase II), consistently copurified with an approximately 50 kDa protein (G Beta h) which is dissociated from G alpha h upon activation with GTP gamma S or AlF4-. Present immunological and biochemical studies on the regulation of the GTPase cycle of G alpha h, which involves the alpha 1-adrenoceptor and 50 KDa G beta h, reveal that the 50 kDa protein is indeed a G alpha h-associated protein and down regulates functions of G alpha h. Thus, polyclonal antibody against G Beta h coimmunoprecipitates GDP-bound G alpha h but not the GDP-AlF4--bound form. The GTP gamma S binding and GTPase activity of G alpha h are inhibited in a G beta h concentration dependent manner. Supporting this notion, G beta h accelerated GTP gamma S release from G alpha h and changes the affinity of G alpha h from GTP to GDP. Moreover, the ternary complex preparation exhibits TGase activity that is inhibited in the presence of the alpha 1-agonist and GTP. The GTP gamma S binding by the ternary complex, consisting of the alpha 1-agonist, the receptor, and Gh, is also inhibited by G beta h. The inhibition of GTP gamma S binding with the ternary complex requires a > or = 2.7-fold higher concentration of G beta h than the G alpha h alone, indicating that the receptor enhances the affinity of G alpha h for GTP. In addition, G beta h copurifies with an alpha 1-agonist, adrenoceptor, and G alpha h ternary complex, showing that the complex is a heterotetramer. Our data also suggest that G beta h does not directly interact with alpha 1-adrenoceptor. These findings clearly demonstrate that G alpha h associates with a novel protein which modulates the affinity of G alpha h for guanine nucleotides and that the GDP-bound Gh is the ground state for the counterpart activator, the alpha 1-adrenoceptor, in this signaling system.

Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B.

N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), serine protease inhibitors, block many cytotoxic functions of immune cells including superoxide anion production, cytokine release, cell-mediated cytolysis, and nitric oxide (NO)-related macrophage functions. IFN-gamma/LPS-induced NO production from murine peritoneal macrophages was inhibited by TPCK and TLCK in a dose-dependent manner (EC50s: approximately 20 microM for TPCK and approximately 30 microM for TLCK). Viability exceeded 91% with 25 microM TPCK and with 80 microM TLCK. When TPCK treatment was delayed until 1 h of activation, the inhibitory effect was markedly reduced. After 2 h of the activation, TPCK was not effective anymore. Addition of either TNF-alpha or conditioned media from IFN-gamma/LPS-activated macrophage culture did not prevent the inhibitory effect of TPCK. Neither TPCK nor TLCK reduced enzymatic NO production from macrophage lysates. Lysates from TPCK-treated cells did not generate NO even after supplementing necessary cofactors for NO synthase. Immunoblotting analysis showed that simultaneous treatment of TPCK with IFN-gamma/LPS abolished the NO synthase expression, whereas delayed addition of TPCK was either partially effective or not effective at all. Furthermore, TPCK treatment reduced the concentration of mRNA for NO synthase without decreasing mRNA stability. Thus, the serine protease inhibitors directly blocked an early event in expression of NO synthase. Electrophoretic mobility shift assay indicated that TPCK blocked the activation of nuclear factor-kappa B, a transcription factor necessary for NO synthase induction. TPCK also blocked disappearance of I kappa B from cytosolic fraction, and nuclear translocation of NF-kappa B subunits p50 and p65. Delaying the addition of TPCK by 10 min partially prevented the inhibition of the NF-kappa B activation process and allowed partial resuming of NO production. Thus, TPCK inhibited NO synthase induction by blocking NF-kappa B activation.

Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function.

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.

Evidence that the Gh protein is a signal mediator from alpha 1-adrenoceptor to a phospholipase C. I. Identification of alpha 1-adrenoceptor-coupled Gh family and purification of Gh7 from bovine heart.

Our previous studies on alpha 1-adrenoceptor-mediated signaling suggested that Gh is a signal mediator. Gh consists of a 74-kDa GTP-binding alpha-subunit and a 50-kDa beta-subunit. Studies using the alpha 1-agonist-receptor-G-protein ternary complexes from various tissues and species revealed that the intensity (GTP-binding) of the [alpha-32P]GTP-labeled proteins resulting from activating the alpha 1-receptor was significantly attenuated by phentolamine. The molecular masses of GTP-binding proteins were 74 kDa in rat heart and liver, 77 kDa in dog heart, 78 kDa (Gh7 alpha) in bovine heart and liver, and 80 kDa in human heart. Supporting these observations, a specific antibody to Gh7 alpha not only recognized these GTP-binding proteins in the ternary complex preparations, but also co-immunoprecipitated alpha 1-adrenoceptors, indicating a tight association of these GTP-binding proteins with the alpha 1-adrenoceptor. These results also demonstrate that functional and structural similarities exist among these GTP-binding proteins. Additionally, one of the identified G-proteins (termed Gh7) was purified from bovine heart. Gh7 consisted of the 78-kDa GTP-binding protein and a 50-kDa protein.

Evidence that the Gh protein is a signal mediator from alpha 1-adrenoceptor to a phospholipase C. II. Purification and characterization of a Gh-coupled 69-kDa phospholipase C and reconstitution of alpha 1-adrenoceptor, Gh family, and phospholipase C.

Our studies on the alpha 1-adrenoceptor signaling have demonstrated that the Gh family is a signal mediator. We report here that a 69-kDa phospholipase C (PLC) is the effector in this signal pathway. The enzyme was isolated by dissociating a Gh7-PLC complex which was induced in the bovine liver membranes incubating with (-)-epinephrine and GTP. The enzyme displayed a marked preference hydrolysis for phosphatidylinositol 4,5-bisphosphate over other phosphatidylinositides at micromolar calcium. Reconstitution of PLC with the alpha 1-adrenoceptor and Gh (Gh7) into phospholipid vesicles resulted in a lowered Ca2+ requirement for the substrate hydrolysis in the presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) when the receptor was activated with the alpha 1-agonist. The formation of inositol phosphate was hormone concentration dependent and reached maximal within 3 min which was faster than the formation in the presence of the alpha 1-antagonist. An Gh7 alpha antibody co-immunoprecipitated 80-85% of phospholipase C activity in the presence of GTP gamma S, but not in the presence of GDP or buffer, showing the association of PLC with the alpha-subunit of Gh family. Thus, our novel approaches to identify the effector involved in the alpha 1-adrenoceptor signaling, as well as the reconstitution studies, substantially demonstrate that the alpha 1-adrenoceptor-mediated transmembrane signaling involves the Gh family and a 69-kDa PLC.

Macrophage nitric oxide synthase subunits. Purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme.

The cytokine-induced nitric oxide synthase (NOS) of macrophages is a homodimeric enzyme that contains iron protoporphorin IX (heme), FAD, FMN, tetrahydrobiopterin, and calmodulin. To investigate how the enzyme's quaternary structure relates to its catalytic activity and binding of prosthetic groups, dimeric NOS and its subunits were purified separately and their composition and catalytic properties compared. In contrast to dimeric NOS, purified subunits did not synthesize NO or contain bound heme or tetrahydrobiopterin. However, the subunits did contain FAD, FMN, and calmodulin in amounts comparable with dimeric NOS, displayed the light absorbance spectrum of an FAD- and FMN-containing flavoprotein, and generated an air-stable flavin semiquinone radical upon reduction of their ferricyanide-oxidized form. Dimeric NOS and NOS subunits were equivalent in catalyzing electron transfer from NADPH to cytochrome c, dichlorophenolindophenol, or ferricyanide at rates that were 8-30-fold faster than the maximal rate of NO synthesis by dimeric NOS. Reconstitution of subunit NO synthesis required their incubation with L-arginine, tetrahydrobiopterin, and stoichiometric amounts of heme and correlated with formation of a proportional amount of dimeric NOS in all cases. The dimeric NOS reconstituted from its subunits contained 0.9 heme and 0.44 tetrahydrobiopterin bound per subunit and had the spectral and catalytic properties of native dimeric NOS. Thus, NOS subunits are NADPH-dependent reductases that acquire the capacity to synthesize NO only through their dimerization and binding of heme and tetrahydrobiopterin. The ability of heme, tetrahydrobiopterin, and L-arginine to promote subunit dimerization is unprecedented and suggests novel roles for these molecules in forming and stabilizing the active dimeric NOS.